Exposure of animals to heavy metal ions, can lead to long term adverse effects such as carcinogenesis and teratogenesis. The mechanisms by which heavy metals can affect gene expression are virtually unknown. We propose to study in detail one well defined effect of exposure to heavy metals: the induction of metallothioneins (MT). These are low molecular weight, cystein rich, heavy metal binding proteins. The human MT gene family was chosen as a model system to study the molecular interactions of both essential and toxic trace metal ions with the human genetic apparatus. Potential environmental pollutants such as cadmium and mercury are potent inducers of MT. As a first step toward elucidating the regulatory mechanisms involved in MT gene expression, we plan to determine the full nucleotide sequence of the various human MT genes which we have recently isolated. The genomic arrangement and linkage of the genes will be studied using "gene walking" techniques and analysis of different mouse x human somatic cell hybrids for presence and expression of human MT genes. A number of cell lines derived from different tissues, will be analyzed for any possible tissue specific differences in MT gene expression and regulation. The chromatin structure of the genes will be studied in various cell lines, differing in their expression and regulation of MT genes. Sensitivity to nucleases such as DNase I and S1 will be used as structural probes. We also plan to examine the effects of exposure to heavy metal ions on the chromatin structure of the MT genes. The nucleotide sequences essential for heavy metal and glucocorticoid induction of the human MT genes will be determined by a combination of in vitro mutagenesis and gene transfer experiments. A series of progressive 5 feet and 3 feet deletion mutations would be analyzed for effects on transcriptional activity and regulation of a MT promoter. Understanding how MT gene expression is regulated might allow us to design more effective countermeasures against heavy metal pollution.